Impact of ATM and SLC22A1 Polymorphisms on Therapeutic Response to Metformin in Iranian Diabetic Patients

Metabolic syndrome and its pathological sequel, type 2 diabetes are considered as important global health problems. Metformin is the most common drug prescribed for patients with this disorder. Consequently, understanding the genetic pathways involved in pharmacokinetics and pharmacodynamics of this drug can have a considerable effect on the personalized treatment of type 2 diabetes. In this study, we evaluated the association between rs11212617 polymorphism of ATM gene and rs628031 of SLC22A1 gene with response to treatment in newly diagnosed type 2 diabetes patients. We genotyped rs11212617 and rs628031 polymorphism by PCR based restriction fragment length polymorphism (RFLP) and assessed the role of this polymorphisms on response to treatment in 140 patients who have been recently diagnosed with type 2 diabetes and were under monotherapy with metformin for 6 months. Response to metformin was defined by HbA1c and fasting blood sugar (FBS) values. Based on such evaluations, patients were divided into two groups: responders (n= 63) and non-responders (n= 77). No significant association was found between these polymorphisms and response to treatment (OR= 0.86, [95% CI 0.52–1.41], P= 0.32) for rs11212617 and (OR= 0.45, [95% CI 0.64–1.76], P= 0.45) for rs 628031. The reported gene variants in ATM and SLC22A1 are not significantly associated with metformin treatment response in type 2 diabetic patients in an Iranian population.

The prevalence of T2D in Asia has increased considerably. International Diabetes Federation data suggest that about 30-60% increase will occur in the prevalence of T2D in many Asian-Pacific countries by the year 2025 (3). In addition, T2D has affected younger population in Asia compared with the Western countries (4). The pathophysiology of T2D is complex. Both βcell dysfunction and insulin resistance contribute in this disorder with abdominal obesity being a major risk factor for the latter (3,5). Hyperglycemia, insulin resistance, and hyperinsulinemia have been shown to contribute to increased risk for many malignancies in diabetic patients (6,7). Accordingly, lifestyle interventions and pharmacotherapy are required to achieve and maintain optimal glucose control and prevent disease related complications (1).
Metformin (1, 1-dimethylbiguanide) is the first-choice and the most widely used drug for treatment of T2D because of its effective, reasonable price and safety (8,9). Its hypoglycemic mechanisms include reduction of hepatic glucose output, partly via reduced gluconeogenesis, decrease in insulin resistance, especially in liver and skeletal tissue, up-regulation of glucose uptake in adipose tissue, and suppression of the intestinal glucose absorption (3,6,10). It also reduces plasma lipid (10). This drug has been used in the treatment of nonalcoholic fatty liver disease, polycystic ovarian syndrome (PCOS), premature puberty as well as prevention of cancer (2-4, 6, 11). It has been shown to influence classical cardiovascular risk factors including LDL-cholesterol, anthropometric indices and blood pressures as well as atherogenic dyslipidaemia, inflammation and vascular function.
Furthermore, it improves haemostasis via reduction of factor VII and Factor XIII levels (3) .Additionally, recent studies have indicated an antioxidant effect for metformin (12).
Consequently, this drug is an attractive modality for treatment of T2D. However, not all patients benefit from metformin since the hypoglycemic response is not seen in a proportion of patients. Furthermore, gastrointestinal side effects make this drug intolerable in a subset of patients (2). Although variation in response to a certain drug can be attributed to drug-drug interactions, age, organ function, simultaneous therapy, the role of genetic factors in variability in drug effects is significant (13). Metformin has been shown to be actively absorbed from the gut and eliminated unchanged in the urine (2). It is transported into the hepatic cells by organic cation transporter1 (OCT)1 (encoded by SLC22A1) (14,15), and into the renal tubules by OCT2 (encoded by SLC22A2) (2,16). OCT1 has been shown to play a significant role in the efficacy of metformin (4). Population studies have shown a high level of polymorphisms for OCT1 in different ethnicities (2). Functional polymorphisms in the corresponding gene such as rs628031 (Met408Val) have been shown to affect its liver uptake, and consequently influence its efficacy (7,17). In addition, ATM (ataxia telangiectasia mutated) is a gene whose role in DNA repair and cell cycle control is evident. It has also been shown to play a significant role in the modulation of metformin effects, and variations in this gene change the response to this drug (8,18,19).
Consequently, in this study we aimed at analysis the association between rs11212617 polymorphism of ATM and rs628031 of SLC22A1 genes and glycemic response to metformin in an Iranian population of diabetic patients. Based on the response to metformin, patients were classified into two groups: responder group (who showed a decrease in HbA1c levels by at least1% from the baseline) and non-responder group.

Laboratory analyses
The

Results
In this monotherapy study, the subjects were split into two groups: responders (n= 63) and nonresponders (n =77). The groups did not differ significantly in age (53.  Table 1. As shown in Table 1, there was a statistically significant difference between responders and non-responders after metformin therapy with respect to systolic blood pressure (SBP) and diastolic blood pressure (DBP). The allele frequencies and genotypes distribution of ATM-rs11212617 and SLC22A1-rs628031 polymorphisms are shown in Table 2 and 3, respectively.   Table 3. Genotypes and alleles frequencies of SLC22A1-rs628031

Discussion
In this study, we have defined the response to metformin in 140 patients with T2D. Contrary to our hypothesis, no association was found between variants in SLC22A1 and ATM genes, and glycemic response to metformin.
There is a huge clinical variation in response to metformin, and this drug is usually combined with other agents such as sulfonylureas to treat diabetes. Clinical trials data have indicated that more than one third of patients receiving metformin monotherapy do not achieve acceptable control of fasting glucose levels (22). The main reason for the lack of a dramatic response in the treatment of these patients may be a variation of genes involved in pharmacokinetics and pharmacodynamics of the drug (2,23,24).
It has been demonstrated that the OCTs and ATM proteins play a dominant role in glycemic response to metformin (14,15,18).
HbA1c level has been assessed as a marker of treatment response in diabetic patients in many studies. In some of them, treatment success has been defined as the ability to reach the treatment target of an HbA1c≤ 7 % (8,18,19). However, Shikata et al. selected reduction of HbA1c values by more than 0.5 % as a cut off point for dividing patients into responders and non-responders (17).  (18). A previous study conducted in T2D patients in the Netherlands and the UK has identified rs11212617 as the first robustly replicated common susceptibility locus associated with metformin treatment response (19).
However, the function of this gene variant is not elucidated so far (25).
Although OCT1 encoded by SLC22A1 gene has been shown to play a significant role in the efficacy of metformin, the association of rs628031 variant with glycemic response has not been assessed before. So, our study is the first study in this regard which shows no significant association between rs628031 alleles and metformin response.
In the present study, the frequency of the mutant allele of SLC22A1-rs628031(M408V) variant between responders and non-responders was not significantly different. The M408V variant has been associated with gastrointestinal side effects in 246 metformin users. A significant lower average HbA1c level in the presence of a lower average dose of metformin in those cases implies a possible relationship between better response to metformin and susceptibility to side effects (26). The local increase of drug concentration in the intestinal tissue is proposed as a mechanism of metformin intolerance. As SLC22A1 and SLC22A3 are also expressed in enterocytes, it has been suggested that genetic variants in these genes may also affect the intestinal metformin uptake and consequently induce gastrointestinal side effects (14).
Previous studies showed that OAD drugs